1. Field of the Invention
The present invention relates to a process for improving the production of methionine by culturing a microorganism modified for enhancing the expression of genes involved in succinate dehydrogenase synthesis. The microorganisms were modified in a way that the methionine/carbon source yield is increased. The isolation of methionine from the fermentation medium is also claimed.
2. Description of Related Art
Succinate dehydrogenase (succinate oxidoreductase, SQR) is a functional member of both the Krebs cycle and the aerobic respiratory chain. SQR catalyzes the oxidation of succinate to fumarate in the bacterial cytoplasm with the concomitant reduction of ubiquinone in the membrane. In E. coli and other bacteria the enzyme is comprised of four subunits. The two hydrophobic subunits, SdhC and SdhD, anchor two hydrophilic and catalytic subunits SdhA and SdhB to the surface of the inner membrane. Five unique cofactors are involved in the oxidation of succinate to fumarate. In SdhA a covalently attached flavin adenine dinucleotide (FAD) molecule is present to catalyze succinate oxidation by a hydride transfer mechanisms. Electrons are then transferred individually through the electron transfer subunit (SdhB) which contains a [2Fe-2S], a [4Fe-4S] and a [3Fe-4S] cluster. A quinone binding site is formed by residues from SdhC, SdhD and SdhB permitting the reduction of ubiquinone to ubiquinole. The enzyme contains also a heme b molecule sandwiched between SdhC and SdhD that is not essential to enzyme function (Yankovskaya et al. 2003, Science 299, 700; Cheng et al. 2008 Biochemistry 47, 6107).
The amino acid L-methionine is an important feed-additive that is produced in large quantities (600000 t/an). Production relies exclusively on chemical biosynthesis and requires crude oil derived precursors. With increasing pressure on the price of these non-renewable resources a sustainable process receives increasing interest. Fermentative production of methionine has thus become an economically viable alternative to the chemical process.
Methionine production by fermentation requires modification of several precursor-providing pathways. Three majors routes contribute to methionine biosynthesis. Aspartate serves as the precursor of the carbon skeleton, cysteine as sulphur donor and methylene-THF as donor of the terminal methyl group. In addition, activation of aspartate-derived homoserine by succinyl-CoA is required for the first step in methionine biosynthesis. Succinyl-CoA is produced in the Krebs cycle and thus increased expression of Krebs cycle enzymes may increase methionine production. Nevertheless high Krebs cycle activity has been shown to be a disadvantage to amino acid production and reducing the activity of Krebs cycle enzymes, such as isocitrate dehydrogenase, can be beneficial to amino acid production (WO2007017710 Metabolic Explorer, WO2009133063 Evonik Industries).
WO 2009078973 (Glycos Biotechnology) and EP1106684 (Evonik Industries) disclose that the deletion of sdh genes, increases the production of amino acids and other metabolites of industrial interest. It is understood from this art that decreasing expression of sdh genes will lower Krebs cycle activity and thus CO2 production, which in turn should have a positive impact on product yield.
There is a need to increase the yield of methionine being produced form a renewable carbon source. Contrary to the teaching of the prior art, that decreased activity of the succinate dehydrogenase increases product yield, it was found that increased expression of the succinate dehydrogenase enzyme increases methionine/glucose yield.